Zebra fish is an easy to maintain, small tropical fish with transparent embryos that develop outside the mother’s body. Their short generation time (3-4 months), high fertility rate (hundreds of eggs per week), rapid development (most organs develop within first 48 hours post fertilization) and advanced genetic techniques (transgenics, forward and reverse genetics) make them an outstanding model for biomedical research. Furthermore, development of their cartilaginous skeleton occurs by similar mechanisms to that of humans, which means zebra fish are suitable as a model for studies on human skeletal diseases.

Using forward genetic screens, hundreds of zebra fish mutations that affect cartilage morphogenesis and/or differentiation have been identified. We positionally cloned two mutations in exostosin genes: dackel (dak/ext2) and boxer (box/exlt3) (Lee et al., 2004, Neuron. 44: 947-960) and are currently using these to study the development of exostoses. Homozygous dak/ext2 mutants show a similar disorganization of cartilaginous skeleton to that seen in HME tumors i.e. chondrocytes, instead of forming long stacks of flattened cells, form non-polarized clusters of rounded cells.

Cartilage formation and differentiation remains unaffected in dak/ext2 mutants, which suggest that the dak/ext2 phenotype probably results from changes in cell division planes or/and cell movements. In support, electron microscope observations verified the presence of abnormalities in the cytoskeleton of dak/ext2 mutant chondrocytes. Furthermore, malformations of cartilage similar to those seen in dak/ext2 are also present in another zebrafish mutant called pipetail (ppt/wnt5a) that is involved in the non-canonical Wnt/Ca2+ planar polarity pathway.

Interestingly, both dak and ppt mutants show a significant delay in endochondrial ossification whereas membranous bones are formed normally. The similarities between these two mutants suggest Ext2 may act through non-canonical Wnt signaling.

In addition to the exostoses-like phenotype, dak/ext2 and box/exlt3 also share other developmental defects (missorted retinotectal projections and malformed pectoral fins) and both of these mutants have significantly reduced level of heparan sulfate proteoglycans (HSPGs). A third mutant, called pinscher (pic), also has the mentioned above defects, suggesting that pic is in the same genetic pathway.

We have recently positionally cloned pic and shown it does not belong the to exostosin gene family. This strengthens the possibility that non-Ext1/Ext2-related cases of HME might be due to mutations in other genes involved in the synthesis of HSPGs.

This work is supported by Wellcome Trust and Cancer Research UK.

The MHE Research Foundation would like to acknowledge and thank Dr. Karlstrom, Biology Department,University of Massachusetts and Dr. Kane, Dept of Biology, University of Rochester for the use of the zebrafish video.

1 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom2 Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom3 Abteilung Genetik, MPI für Entwicklungsbiologie, Tuebingen, Germany4 Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America5 Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United States of America6 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, United States of America7 Brain Institute, University of Utah, Salt Lake City, Utah, United States of AmericaAbstract

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak−/− cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic−/− chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.

The MHE Research Foundation would like to extend its appreciation to Dr. Henry Roehl who is a member of our foundation's Scientific and Medical Advisory Board and to Dr. Malgorzata Wiweger for all of their continuing, dedicated research efforts on behalf of all people affected by MHE/MO/HME

The presence of dental problems has been communicated to us by some MO patients, but as far as we know, there are no medical data published that would confirm this observation. As problems with teeth might severely affect not only the self esteem but also general health, hence we recognize that dental health in MO patients needs to be investigated.

The MHE Research Foundation with the support and collaboration of Pancras Hogendoorn, M.D., Ph.D. and Malgorzata Wiweger, Ph.D. from the Leiden University Medical Center(LUMC) The Netherlands a questionnaire has been designed that could be answered without need of seeing any dentist or other medical professionals. We hope that this survey will contribute to better understanding of the MO disorder and improve the quality of care given to MO patients.

The research team aims to make the results publicly available in a form of an article published in a scientific journal, so basic researchers; medical professionals and people affected by MO will be able to read the results.

Multiple Osteochondromas (MO; previously known as HME/MHE) is characterized by the formation of cartilaginous bone tumors (osteochondromas) at multiple sites in the skeleton, bone curving, short stature, bursa formation and impingement of nerves, tendons and vessels. MO is also known to be associated with arthritis, general pain, scarring, increased risk of bone fracture and malignant transformation. MO patients present additional complains but the relevance of those needs validation. We use a zebrafish mutant called dackel (dak) as a model for MO. dak is mutated in a homologue of the human EXT2 gene, and is the only Heparan Sulfate-deficient vertebrate being available that can complete embryo development when homozygous for a mutation in an ext gene. The dak (-/-) mutant displays such severe phenotypes that the identification of more settled defects is thus possible. Histological analysis revealed that the dak (-/-) mutant has very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of homozygote dak/ext2 mutants had a single tooth formed at the end of the 5th pharyngeal arch, whereas wild-type fish had developed three teeth, located in the middle of the pharyngeal arch. dak (-/-) teeth had abnormal morphology (they were shorter and thicker than in the WT) and scattered ossification at the tooth base. Deformities such as spitted crowns and enamel lesions were found in 20% of the heterozygote dak/ext2 adults. The dak-tooth morphology was partially rescued by a treatment with either FGFs or TGF that was added into fish water. The number of teeth was not affected unless a higher dosage of FGF8 was admitted locally. In order to validate our findings in zebrafish, a dental questionnaire was designed and sent out to the HME Research Foundation for further distribution among MO patients and their families. Until now, we have received 23 replies from MO patients, half of whom stated that they have malformed and/or displaced teeth with abnormal enamel; further indicating that MO might indeed be also associated with dental problems. The analysis of dak skeleton also revealed severe bone defects in all homozygote larvae and in 20% of heterozygote adult dak/ext2 mutants. Bone malformations in dak/ext2 mutants coincided with reduced expression of osteoblast markers and enhanced TRAP staining. This indicates that the remodeling of the skeleton might be altered in MO, and so patients might be at higher risk of developing osteoporosis. Skeletal and dental defects, similar to those present in dak/ext2 mutant, were also observed in the acerebellar (ace/fgf8) zebrafish mutants. This observation suggests that FGF signaling might be a potential target for prophylactics in MO.

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